John M. Rawls

The enzymes for de novo pyrimidine biosynthesis in Drosophila melanogaster: Their localization, properties and expression during development☆

Abstract 1. 1. The first three and last two pyrimidine pathway enzymes are soluble, cytoplasmic activities in Drosophila melanogaster larvae and evidence is presented showing that the last two enzymes comprise a bifunctional enzyme complex. 2. 2. The fourth pathway enzyme, dihydroorotate dehydrogenase, is a particulate activity that co-sediments with mitochondrial cytochrome oxidase. 3. 3. During development, the soluble enzymes exhibit maximal values in embryos and young larvae and show minimal values in later larval stages, pupae and adult males: adult females show high activities due to oocytes and retained embryos. 4. 4. Mutations of the rudimentary locus destroy only the initial three pathway enzymes.

Expression of the dihydroorotate dehydrogenase gene, dhod, during spermatogenesis in Drosophila melanogaster

The dhod gene encodes dihydroorotate dehydrogenase (DHOdehase), which catalyzes the fourth step of de novo pyrimidine biosynthesis. In addition to the common 1.5 kb dhod RNA expressed by embryos and females, adult males produce a group of slightly longer RNAs. Evidence is presented that the latter RNAs arise through transcription initiation at sites upstream from that of the common RNA and expression of these male-specific RNAs is limited to spermatogenesis. In situ hybridization analysis shows that these RNAs accumulate during spermatocyte growth and persist through meiosis and early spermatid differentiation. In contrast, DHOdehase activity is virtually absent in spermatocytes, meiotic cells, and in early spermatid cysts, then it becomes highly abundant in elongated spermatid cysts and disappears in late spermatogenesis. Thus, testis-limited expression of dhod conforms to a model proposed for other genes that function during spermiogenesis : transcription in spermatocytes, storage of translationally inactive RNA through meiosis, translation of the RNA during spermiogenesis. Very similar expression of a testis promoter-lacZ fusion transgene indicates that sequences required for the spermatogenesis transcription and translation patterns are confined to the 5′ end of the dhod gene. Deletion analysis of that 5′ region delimits all sequences necessary for spermatid expression of the transgene to a 89 by fragment. These results are discussed in the contexts of known mechanisms of gene regulation during spermatogenesis and potential roles of DHOdehase during spermiogenesis.

Analysis of Pyrimidine Catabolism in Drosophila melanogaster Using Epistatic Interactions With Mutations of Pyrimidine Biosynthesis and β-Alanine Metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with β-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; β-alanine synthase (βAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (rSu(b)) of the dark cuticle phenotype of black mutants in which β-alanine pools are diminished; these results confirm that pyrimidines are the major source of β-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.

Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster.

Flies mutant for one or both of the last two enzymes of de novo pyrimidine biosynthesis express a number of phenotypes that are also expressed by mutants of the first four pathway enzymes (r and Dhod-null mutants). However, r-1 flies also express two phenotypes, mottled eyes and poor viability, that are not usually expressed by r and Dhod-null flies. Chemical determinations show that orotic acid, a substrate for the fifth pathway enzyme, accumulates in r-1 individuals but not in r and wild-type individuals. Moreover, flies simultaneously mutant for r and r-1 do not express the mottled-eye phenotype, showing that r is epistatic to r-1 for this r-1-specific phenotype. When genotypically wild-type flies are cultured on a medium containing 6-azauracil, the base of a potent inhibitor of the last enzyme of de novo pyrimidine biosynthesis, phenocopies are obtained that include the mottled-eye as well as the wing phenotypes of r-1 flies. These results support hypotheses that the phenotypes common to r, Dhod-null, and r-1 flies are consequences of uridylic acid deficiency, whereas the r-1-specific phenotypes result from orotic acid accumulation in flies lacking either or both of the last two enzymes of de novo pyrimidine biosynthesis.

Drosophila melanogaster dihydroorotate dehydrogenase: the N-terminus is important for biological function in vivo but not for catalytic properties in vitro

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis, is an integral flavoprotein of the inner mitchondrial membrane and is functionally connected to the respiratory chain. Here, experiments have been directed toward determining the roles of the N-terminal sequence motifs both in enzymatic properties of insect DHODH produced in vitro and the in vivo function of the protein. Full-length and three N-terminal truncated derivatives of the Drosophila melanogaster enzyme were expressed in Escherichia coli and purified. For identification on Western blots of recombinant DHODH as well as the native enzyme from flies polyclonal anti-DHODH immunoglobulins were generated and affinity-purified. The enzymatic characteristics of the four versions of DHODH were very similar, indicating that the N-terminus of the enzyme does not influence its catalytic function or its susceptibility to prominent DHODH inhibitors: A77-1726, brequinar, dichloroallyl-lawsone and redoxal. Whereas the efficacy of A77-1726 and dichloroallyl-lawsone were similar with Drosophila and human DHODH, that of brequinar and redoxal differed significantly. The differences in responses of insect DHODH and the enzyme from other species may allow the design of new agents that will selectively control insect growth, due to pyrimidine nucleotide limitation. In vivo expression of the full-length and N-truncated DHODHs from engineered transgenes revealed that the truncated proteins could not support normal de novo pyrimidine biosynthesis during development of the fly (i.e., failure to complement dhod-null mutations), apparently due to instability of the truncated proteins. It is concluded that the proper intracellular localization, directed by the N-terminal targeting and transmembrane motifs, is required for stability and subsequent proper biological function in vivo.

Molecular cloning of the UMP synthase gene rudimentary-like from Drosophila melanogaster

SummaryThe rudimentary-like locus encodes UMP synthase, a bienzyme protein containing the last two enzyme activities of de novo pyrimidine biosynthesis: orotate phosphoribosyltransferase and orotidylate decarboxylase. This locus lies within chromosome region 9313. New mutations have been used to refine the 9313 cytogenetic map and a chromosome walk has been executed to clone DNA from this region. DNA encoding UMP synthase was identified using mixed oligonucleotides which were based on sequences derived from conserved peptide tracts of the protein in other species. cDNA clones of the embryonic UMP synthase mRNA have been isolated and used to define the extent of genomic DNA sequences which encode the transcript. The embryonic RNA is approximately 1.75 kb in length.

Organization of transcription units around the Drosophila melanogaster rudimentary locus and temporal pattern of expression

SummaryThe molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlated well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females.

The Dhod locus of Drosophila: Mutations and interrelationships with other loci controlling de novo pyrimidine biosynthesis

SummaryMutations at the Dhod locus have been isolated following ethylmethanesulfonate mutagenesis. These mutants express those phenotypes common to other mutations of the de novo pyrimidine pathway: specific wing and leg defects and female sterility. Dihydroorotate dehydrogenase activity is severely reduced in all Dhod mutants, whereas levels of the other pathway enzymes are largely unaffected. The twelve Dhod mutations described here comprise a single complementation group. All of these mutations are nonlethal and the collection includes apparent amorphic as well as hypomorphic alleles. These results are discussed relative to the properties of the complex loci that encode the other steps of de novo pyrimidine biosynthesis.

Divergent Functions Through Alternative Splicing: The Drosophila CRMP Gene in Pyrimidine Metabolism, Brain, and Behavior

DHP and CRMP proteins comprise a family of structurally similar proteins that perform divergent functions, DHP in pyrimidine catabolism in most organisms and CRMP in neuronal dynamics in animals. In vertebrates, one DHP and five CRMP proteins are products of six genes; however, Drosophila melanogaster has a single CRMP gene that encodes one DHP and one CRMP protein through tissue-specific, alternative splicing of a pair of paralogous exons. The proteins derived from the fly gene are identical over 90% of their lengths, suggesting that unique, novel functions of these proteins derive from the segment corresponding to the paralogous exons. Functional homologies of the Drosophila and mammalian CRMP proteins are revealed by several types of evidence. Loss-of-function CRMP mutation modifies both Ras and Rac misexpression phenotypes during fly eye development in a manner that is consistent with the roles of CRMP in Ras and Rac signaling pathways in mammalian neurons. In both mice and flies, CRMP mutation impairs learning and memory. CRMP mutant flies are defective in circadian activity rhythm. Thus, DHP and CRMP proteins are derived by different processes in flies (tissue-specific, alternative splicing of paralogous exons of a single gene) and vertebrates (tissue-specific expression of different genes), indicating that diverse genetic mechanisms have mediated the evolution of this protein family in animals.